CA2190367A1 - Isolation of hematopoietic dendritic cells by high gradient magnetic cell sorting - Google Patents

Abstract

Methods are provided for the rapid isolation of highly purified and functionally intact dendritic cells from a mixed cell population, using colloidal superparamagnetic particles. Dendritic cells are enriched from a blood or lymph sample using a two step high-gradient magnetic cell separation. Lymphocytes, natural killer and monocytic cells are depleted by specific binding to markers present on lymphoid and myeloid cells. In a separate step, dendritic cells are enriched by HGMS. Purified dendritic cells are useful as a source of antigen presenting cells for in vitro analysis, and in immunomodulating therapies, particularly for priming naive T cells.

Description

~ ~096/28732 2 ~ 90367 r~ . M~
s ISOLAllON OF HEMA I OPO~ DENDRITIC CELLS BY HIGH GRADIENT
MAGNETIC CELL SORTING
INTRODUCTION
Technical Field The field of this invention is the isolation of h~ oi~ . dendritic cells.
Backgrsund The role and identity of antigen ~,~s~"~i"g cells has been the subject of controYersy in the past. Both mal,,o~l,ages and dendritic cells (sometimes 20 referred to as Iymphoid i"le,~iyi~dli"g dendritic cells) are now known to present antigen. Ma~,o~ dyes present to activated T cells and B cells. However, T
helper cell priming is depel1de"l on antigen ~ e"ldlk)" by dendritic cells. It appears that dendritic cells pick up and process antigen in the peripheral blood, then travel into the Iymph, and finish maturation in the pdld~Ulli~,dl T cell 2s zone of Iymph nodes. Md~,,u~ llages and dendritic cells at all levels of maturity can present antigen to preactivated T cells, but only mature dendritic cells areable to prime naive T cells.
Dendritic cells are derived from h~"~ .l,oiæ~i.. stem cells in the bone marrow. Precursor and immature dendritic cells are found in the blood and 30 Iymph. The ",orl,llolo~ic~'ly distinct, fully mature dendritic cells are found in the spleen and Iymph node. In keeping with their role in antigen ~r~s~"ldli~", dendritic cells express high levels of MHC class I and class ll proteins.
A number of methods have been described for the isolation of dendritic cells from peripheral blood mononuclear cells. A common denominator of 3s these techniques is the requirement for an in vitro culture period of 1-2 days.
During this time, dendritic cells acquire a low bouyant density and little or noadherence to plastic, features that aid in their s~JdldliOIl from other cells.

2 l 9 0 3 6 7 Recently, procedures have been described to isolate dendritic cells from fresh blood. These methods all require a T cell depletion step, followed by flowcytometric sorting. However, the high l~ lolo~k,dl effort required for FACS
has prevented its routine use. FACS sortin3 is a time consuming and cost s intensive procedure. FACS sorting has the additional disadvantage in that it is difficult to sort large numbers of cells, or to sort multiple samples at the same time.
An alternative approach to cell sorting has been described, whereby magnetic mi~,,upa~ ,les coupled to d"li~odies are used to select for specific cell types. An improved sepd,dli~" process whereby dendritic cells could be sorted from blood, and which allows multiple, and potentially large, samples to be run on the bench would provide numerous benefits in the characterization and use of antigen ~ sel,lillg dendritic cells.
Relevant Literature The isolation of dendritic cells after a period of time in culture is described in Cameron et al. (1992) Science 257:383; Langhoff et al. (1991) P.N.A.S. 88:7998; Chehimi et al. (1993) J. Gen. Virol. 74:1277; Cameron et al.
(1992) Clin. FYr Immunol. 88:226; Thomas etal. (1993) J. Immunol. 150:821;
and Karhumaki etal. (1993) Clin. EYn. Immunol. 91:482.
The isolation of dendritic cells from peripheral blood by flow cytometric sorting is described by Thomas et al. (1994) J. Immunol. 153:4016; Ferbas et al. (1994) J. Immunol. 152:4649; and O'Doherty etal. (1994) Immunolo~y 82:487.
2s Activation of naive T cells by antigen pulsed dendritic cells is described in Flamand et al. (1994) Eur. J. Immunol. 24:605-610; Mehta-Damani et al.
(1994) J. ImmunQloav 153:996 and Sornasse et al. (1992) J. Exp. Med. 175:15-21 .
High gradient magnetic cell sorting is described in Miltenyi et al. (1990) Cytometrv 11:231-238. Molday, U.S. 4,452,773 describes the ~ dldli~nl of magnetic iron-dextran microspheres and provides a summary describing the Yarious means of pl~pdldliOI1 of particles suitable for dlld~,lllll~lll to biological materials. A des.,li~,liol~ of polymeric coatings for magnetic particles used inHGMS are found in DE 3720844 (Miltenyi) and Miltenyi et al., U.S. 5,385,707.
Methods to prepare superparamagnetic particles are described in U.S. Patent No. 4,770,183.

WO96/28732 21 90367 r .,~ c SUMMARY OF THE INVFNTION
Methods are provided for the rapid isolation of highly purified and functionally intact dendritic cells from a mixed cell population, usin3 colloidal su~ual~ar~lllagnetic particles. Dendritic cells are enriched from a blood or Iymph sample using a two step high-gradient magnetic cell sepd,dliol1. B-cells, T cells, NK cells and monocytic cells are depleted by specific bindin3 tomarkers present on Iymphoid and myeloid cells. In a separate step, dendritic cells are enriched by HGMS. The isolated dendritic cells are optionally culturedin vitro in the presence of cytokines. Purified dendritic cells are useful as a 0 source of antigen presentin3 cells for in vitro analysis, and for use in immunomodulating therapy, particularly for priming naive T cells.
BRIEF DESCRIPTION OF THF DRAWINGS
Fi3ures 1A(i) to 1C(i) show the clldlduLeri~dlion of peri~ e,dl blood mononuclear cells for HLA-DR and CD3, CD14, CD16 and CD1~ ,u,~s~iun during the seudld~iul~ of dendritic cells. Figures 1A(ii) to 1C(ii) show the side and forward scatter of cell populations durin3 the sepd,dLiun procedure.
Figures 2A and 2B show the ex,uressiol1 of CD4 and CD11c, respectively, by isolated dendritic cells enriched for HLA-DR positive cells.
Figures 2C and 2D show the e~ as;u~) of CD33 and CD11b, respectively, in ullse~udldled peripheral blood mononuclear cells and in isolated dendritic cellsenriched for HLA-DR positive cells.
Figure 3 shows the forward and side scatter of isolated peripheral blood dendritic cells after being subjected to in vitro culture.
Figures 4A(i) to 4C(i) show the chdlduleli~dlion of peripheral blood mononuclear cells for CD4 and CD3, CD14 and CD16 e~wlt:~siun during the sepd~dliol1 of dendritic cells. Figures 4A(ii) to 4C(ii) show the side and forward scatter of cell populations during the sepa,d~iu" procedure.
Figure 5 shows the e,~ ssiu,1 of HLA-DR by isolated dendritic cells enriched for CD4 positive cells.
DESCRIPTION OF THE SPECIFIC EMBODIMFNTS
Methods for the sepa,dli~"" culture and use of he".~ dendritic cells are provided. A blood sample is drawn from a suitable host, and 3s p,~pa,dliul1 made of mononuclear cells from the blood. Dendritic cells are enriched from the mixed blood cell population by a c~"~bil,dLio,1 process involving depletion of non-dendritic cells and enrichment of dendritic cells, WO 96/28~3Z 2 1 9 0 ~ 6 7 using high-sradient magnetic cell s~pdld~iun, or a c~",~i"alio,1 of high and lowgradient magnetic sepa,dlion. A suspension of blood cells are labeled with su,uelludldlllayll~ic particles specific for cell surface antigens, then sorted by bindins to magnetic columns.
The use of high-gradient magnetic cell sorting to enrich for dendritic cells provides several benefits when ~,c,lllpdl~d to flow cytometry methods presently used today. The subject methods require inexpensive reagents and apparatus, which are easily used and maintained. By setting up multiple columns, many samples can be u,ucessed at the same time. An automated system can be used to simplify ~.,ucessi"g of large sample numbers and large volumes.
The subject methods proYide for a highly enriched population of he",.,~ oi-liG dendritic cells and precursors thereof, usually at least about 90% of the population will be dendritic cells or precursors thereof, more usually at least about 95%. The purity may be evaluated by various methods.
Conveniently, flow cytometry may be used in conjunction with light CI~IP.,I,.I.I~
reagents specific for cell surface markers ext~ ssed by dendritic cells. The dendritic cells sepa,d~ed by the subject methods are ~1er"d~upoietic cells ,lldlduleli~ed as ~ ssing class I and class ll MHC proteins, e.g. the human class ll proteins HLA-DP, HLA-DQ and HLA-DR; and class I proteins HLA-A, HLA-B and HLA-C. The dendritic cells also express CD45, CD33 and, for the most part, CD4. The cells lack t:.cp~ssion of most Iymphoid and monocytic speclfic cell markers, e.g. CD3, CD11b, CD14, CD16 and CD19. The mature subset of dendritic cells found in blood are ~,I,a,d~le,i~d by expression of CD1 1c, high levels of CD33, and CD45RO, and are able to present antigen so as to stimulate naive or preactivated T cells. Precursor dendritic cells are CD11c negative, express low levels of CD33, are CD45RA positive, and will differentiate into the mature cells after in vitro culture, as described in somedetail below. The term dendritic cells (DC) shall be intended to include both mature and precursor cells as found in the blood, unless speuiti "y stated otherwise.
Blood sample, as used herein, shall be intended to include hematopoietic biological samples such as blood, Iymph, leukophoresis product, bone marrow and the like; also included in the term are derivatives and fractions of such fluids. The sample may be subjected to prior treatment, such as dilution in buffered medium, wllcerlildlic~ filtration, or other gross treatment that will not involve any specific st:,CdldliUII. The blood sample is W096128732 21 903 67 r~ c drawn from any site, conveniently by venipuncture. The sample is usually at least about 20 ml, more usually at least about 40 ml and may be as large as about 500 ml, more usually not more than about 250 ml. The blood is treated by conventional methods to prevent clotting, such as the addition of EDTA, s heparin or acid-citrate~dextrose solution.
A ~ dldliOI1 of nucleated cells is made from the sample. Any procedure that can separate nucleated cells from erythrocytes is ~ep~ le The use of Ficoll-Paque density gradients or elutriation is well documented in the literature. Alternatively, the blood cells may be resuspended in a solution which selectively Iyses adult erythrocytes, e.g. ammonium chloride-potassium;
ammonium oxalate, etc.
The sample of nucleated peripheral blood cells (NPBC) is selectively depleted of non-dendritic cells. Depletion reagents attached to s~",erl d,d",a~u,"etic particles are bound to cell surface antigens that are present on Iymphoid and monocytic blood cells, but are low or absent on dendritic cells. Especially useful depletion reagents are d"liL,odies against cell surface antigens. Whole a"liuo.3;~ may be used, or fragments, e.g., Fab, F(ab')2, light or heavy chain fragments, etc. Such dl ~iuodies may be polyclonalor Illol~oclul~dl and are generally CCllllllt:l.,;.,"~ available or alternatively, readily produced by techniques known to those skilled in the art. Antibodies selected for use in depletion will have a low level of non-specific staining, and will usually have an affinity of at least about 100 ~LM for the antigen.
Generally, a cocktail of depletion reagents will be used, in order to deplete a wide range of blood cell types. Generally, at least about 75% of the mononuclear peripheral blood cells will be bound by the cocktail of depletion reagents. Suitable antigens for depletion are antigens specific for monocytes, T cells, NK cells and B cells, e.g. CD14 or CD11b, which is found on monocytes; CD3, which is found on T cells; CD16, which is found on NK cells;
and CD19, which is found on B cells. Other useful cell surface antigens include the T cell markers CD2, CD5, CD6 and CD7, the B cell markers CD20, CD21, CD22, CD23, CD24 and CD37, the NK cell and neutrophil marker CD16, also CD56, CD57 and CD94, and the granulocyte marker CD15. In a preferred embodiment, a cocktail of dl,liuo~ies specific for CD3, CD14 or CD11b is used, optionally including CD16 andlor CD19. An alternative combination is dl ~liuodies specific for CD3, CD1 1 b and CD16.
The depletion reagent antibodies are coupled to supel,ua,d,,,ag,,~licparticles, which can be prepared as described in U.S. Patent nos. 4,452,773 WO 96/28732 2 1 9 ~ 3 6 7 PCI/US96/03265 and 4,230,685. The ,,,i.,,u,ud,li-,les will usually be less than about 100 nm indiameter, and usually will be greatcr than about 10 nm in diameter. The exact method for couplin3 is not critical to the practice of the invention, and a number of alternatives are known in the art. Direct coupling attaches the dll~iL,odi~s to 5 the particles, as described in co-pending patent:,, '; ' no. 08/252,112, herein i,,co,,uo,d~ed by reference. Indirect coupling can be accu,,,pli~,,,ed byseveral methods. The depletion rea~ent ar"i~ '-9 may be coupled to one member of a high affinity binding system, e.g. biotin, and the particles attached to the other member, e.g. avidin. One may also use second stage dl ,~iL,odies 10 which recognize species-specific epitopes of the depletion a"~iuodies, e.g.
anti-mouse lg, anti-rat lg, etc. Indirect coupling methods allow the use of a single ",a~"t~ lly coupled entity, e.g. antibody, avidin, etc., with a variety of depletion antibodies.
One preferred method uses hapten-specific second stage a~ odies 15 coupled to the su,uel,udldllla~ iu particles, as described in co-pending patent , no. 08/252,112. The hapten specific dl ,~i~odies will usually have an affinity of at least about 100 ~LM for the hapten. The depletion antibodies are conjugated to the d,u,u,uurid~e hapten. Suitable haptens include digoxin, diuoxi~"i", FITC, dil li~l uphe~ Iyl, nitrophenyl, etc. Methods for conjugation of the 20 hapten to antibody are known in the art.
While not necessary for practice of the subject methods, it may be useful to label the depletion dllli~odits with a fluolu~l"u",e, e.g. phycoerythrin, FITC, rhodamine, Texas red, allophycocyanin, etc. The fluorochrome label may be used to monitor mk,~v:.col ` "y or by flow cytometry the cell co",posili~,1 after 2s the depletion and e",icl""~ steps. Fluorescent labeling may conveniently utilize the same indirect coupling system as the magnetic particles. For example, a cocktail of digu~.ig~"i,l-coupled depletion dllli~odi~s may be used in combination with anti-digu,~iu~"i" antibody coupled to magnetic particles, followed by labeling with a fluorochrome conjugated antibody directed to the 30 anti-hapten antibody.
The depletion reagent dllliuodi~s are added to a suspension of NPBC, and incubated for a period of time sufficient to bind the available cell surfaceantigens. The incubation will usually be at least about 5 minutes and usually less than about 30 minutes. It is desirable to have a sufficient cu"cc:"l,dlion of 3s antibodies in the reaction mixture, so that the efficiency of the magnetic sepa,dliun is not limited by lack of antibody. The auuluulid~ co,~ce"l~d~iol1 isdetermined by titration. The medium in which the cells are separated will be -WO 96128732 2 1 9 0 3 6 7 PcT~us96~0326s any medium which maintains the viability of the cells. A preferred medium is pl~o~l,..~ buffered saline cc",~cli"i,)g from 0.1 to 0.5% BSA. Various media areco",i"e,~ available and may be used according to the nature of the cells, including Dulbecco's Modified Eagle Medium (dMEM), Hank's Basic Salt Solution (HBSS), Dulbecco's pl)O~ dlt~ buffered saline (dPBS), RPMI, Iscove's medium, PBS with 5 mM EDTA, etc., frequently supplemented with fetal calf serum, BSA, HSA, etc Where a second sta3e ,1~au": 'Iy coupled antibody is used, the cell suspension may be washed and resuspended in medium as described above prior to incubation with the second stage dl ,li~o.lies. Al~l, Idli,Icly, the second sta3e antibody may be added directly into the reaction mix. Vvhen directly coupled depletion antibodies are used, the cell suspension may be used directly in the next step, or washed and resuspended in medium.
The suspension of ,,,au,,,:Lics'ly labeled cells is applied to a column or chamber as described in WO 90107380, herein i"co, ~o, d~d by reference. The matrix may consist of closely packed l~u,,,au,,elic spheres, steel wool, wires, ",au"~ic,ally ~pO115i~ fine particles, etc. The matrix is c~",posed of a ferromagnetic material, e.g. iron, steel, etc. and may be coated with an i~"~ "eable coating to prevent the contact of cells with metal. The matrix should have adequate surface area to create sufficient magnetic field gradients in the sepd,dli~" chamber to permit efficient retention of ",au,1." 'Iy labeled cells. The volume necessary for a given s~pd,dliol~ may be empirically determined, and will vary with the cell size, antigen density on the cell surface, cell number, antibody affinity, etc.
In order to maximize the purity of the final cell preparation, a two stringency system is employed, where the depletion step captures a high percentage of labeled cells and the enrichment step captures a lower percentage of labeled cells. This reduces the probability that labeled cells will be carried over from the first sepd, dliul, step into the second. The stringency of the depletion column will be such that at least about 95% of the labeled cells will be retained on the column in the presence of a magnetic field, usually at least about 99% of the labeled cells will be retained, and preferably at least about 99.9% retained. The geometry, matrix co",posilion, magnetic field stren3th, size and flow rate of the ferromagnetic column will determine the percent of labeled cells that are retained on the column. Factors that will - increase the strin3ency are increased column size and len3th, decreased flow rate, and a finer matrix c~lllposiliul1. A column matrix of fibers is preferred for WO 96128732 2 1 9 ~ 3~6 7 ~ .. C
the depletion step. An empirical d~l~,l"i"dliun of the stringency may be made by analysis of bound and unbound cells.
The labeled cells are bound to the matrix in the presence of a magnetic field, usually at least about 100 mT, more usually at about 50û mT, usually not s more than about 2T, more usually not more than about 1T. The source of the magnetic field may be a pe""d"en~ or electromagnet. The unbound cells contained in the eluate are collected as the eluate passes through the column.
For greater purity, the unbound cells may be passed a second time over the magnetic column.
The unbound cells are used in an ~Illk,lllll~llL step, to select for dendritic nucleated cells. Enrichment reagents attached to S~l,Udldllla~ , particles are bound to cell surface antigens that are present on dendritic cells. Of particular interest is the use of reagents specific for MHC class ll proteins, e.~.
HLA-DR, HLA-DQ and HLA-DP or other cell surface markers specir "y present on dendritic cells, such as CD4 for dendritic cells and precursors, or as d~p,uuridle, CD11c for mature dendritic cells. CD45RO may be used to select for mature dendritic cells, and CD45RA to select for precursor cells.
The choice of e",i~,l""~"l reagent will determine to some extent the choice of depletion reagents, based on the distribution of ex,u,~ssir.,~ of the particular markers. The depletion reagents will be selected so as to specifically deplete non-dendritic cells c:~ul~asillg the e,~ ,l""t:"L marker. For example, CD4 is highly expressed by T cells and monocytes, and so the depletion step preceding CD4 selection will include T cell and monocyte specific reagents. MHC class ll proteins are absent or ~,~,u,~ssed at low density on T cells, but are e,.,u,~ssed by B cells and monocytic cells, and so the depletion step preceding HLA class ll selection will include B cell and monocytic cell specific reagents.
Reagents specific for the HLA class ll proteins may be allele specific, i.e.
directed to polymorphic regions of the protein, or directed to conserved sequences. HLA-DR is relatively invariant in sequence, while HLA-DR~ is highly polymorphic. Generally, reagents will be chosen that recognize HLA
proteins from a large number of individuals. However, it may be desirable to isolate dendritic cells having a particular haplotype through the use of allele specific reagents.
3s Conveniently, the enrichment reagent will provide for magnetic labeling through an indirect coupling different from that used for the depletion. The initial binding reaction may combine both e,~,icl""er,~ and depletion reagents.

WO 9612M32 2 1 9 0 3 6 7 . ~ LC
The second stage magnetic particles specific for the enrichment reagent is added after co",ple~iu,1 of the depletion. Alternatively, a directly coupled enrichment rea3ent may be used.
The e"ri.;l"nenL reagents, s~perl,d,d",ay"t:lic particles, columns and s buffers are prepared as described for the depletion reagents, however, the stringency for the e"ri..l""er,l column will be lower than for the depletion column. The stringency of the enrichment column will be such that at least about 50% of the labeled cells will be retained on the column in the presence of a magnetic field, usually at least about 80% of the labeled cells will be 0 retained, usually not more than 95% retained. A column matrix of spheres ispreferred for the enrichment step. The cells are bound to the magnetic matrix.
After the initial binding, the matrix is washed with any suitable physiological buffer to remove unbound cells. The unbound cells are discarded.
The bound cells are released by removing the magnetic field, and eluting in a suitable buffer. The cells may be collected in any d,up,upridle medium which maintains the viability of the cells. Various media are commercially available and may be used according to the nature of the cells, including dMEM, HBSS, dPBS, RPMI, PBS-EDTA, PBS. Iscove's medium, etc., frequently supplemented with fetal calf serum, BSA, HSA, etc.
In many cases the sepd,dlion procedure will perform the depletion step first, followed by the e",i~,l""~"~ step. If the enrichment step is to be perru""ed first, then an additional step is necessary after the en~icl""elll, in order to remove the magnetic label from the enriched cells. This may be accu",,ul;~l,ed by any suitable method. The enriched cell population may be incubated with a 2s solution of d_,~l,d"ase, where the dextranase is present at a co,,ce,,l,dliu,, sufficient to remove suu:,la,~"-"y all mi~"u~,a,li-,les from the labeled cells.
Usually the reaction will be complete in at least about 15 minutes. The depletion step may then be performed as previously described with the dt~ ase treated cells.
Alternatively, the enrichment step may be performed first, and the depletion step modified to use large magnetic spheres in place of the microparticles. The use of such magnetic spheres has been previously described, and the reagents are commercially available. The enriched cell population is incubated with highly magnetic polymer spheres of about 1 to 1û
llm diameter conjugated to the depletion antibody cocktail. The mixture of cellsis then placed in close proximity to a magnetic field. Sub:~ldllli~llJ all cellsbound to the polymer spheres are bound to the magnet within about 1 minute, WO 96128732 2 ~ 9 0 3 6 7 PCT/US96103265 and not more than about 5 minutes. The unbound cells may be decanted and used.
After the depletion and enrichment steps are complete, the cells may be used immediately for antigen ,Ult:s~ dliUI " further analysis of DC function, as a source of mRNA for use in cDNA synthesis, e~c. The mature cells and p,ugen' cells may be compared by cDNA subtraction to determine .lirrt,r~nces in gene e,~,u,l ~io,~ during the maturation process, and to identify specific genes ex,.,, ~ssed by mature dendritic cells.
Alternatively, the cells may be cultured in vitro for a period of time suflicient to induce further maturation, usually at least about 1 day and not more than about 7 days, more usually about 2 to 3 days. The dendritic cells are cultured in an app,op~idL~ liquid nutrient medium, which medium may further comprise one or a combination of cytokines at a col1ce"llGIion sufficient to enhance the dirr~e"Lidlion of precursor dendritic cells into mature antigen ~1~5~1 llil 1~ cells. Cells will be grown at a concentration from about 1û4 per ml to about 1U6 per ml, usually about 1û4 to 1ûs. Various media are CCllllllt:ll 'l~
available and may be used, including Dulbecco's Modified Eagle Medium (dMEM), Hank's Basic Salt Solution (HBSS), Dulbecco's pl,os~ul~dle buffered saline (dPBS), RPMI, Iscove's medium, etc., frequently sl~upl~",~:"l~d with serum, usually heat i"a.,li~ldled normal human serum, generally at a concentration of from about 5-15%, preferably about 10%. Ap~l up~id~e dl~libiulics to prevent bacterial growth and other additives, such as pyruvate (0.1-5 mM), glutamine (0.5 - 5 mM), 2-mercaptoethanol (1 -10 x 10~5 M) may also be included.
2s Cytokines of interest include IL-1, IL-2, IL-3, IL~, GM-CSF and TNF-a.
The addition of GM-CSF to cultures of precursor, i.e. CD1 1c negative, dendriticcells is of particular interest. The factors that are employed may be naturally occurring or synthetic, e.g. prepared l~co,,,bi,,d,,lly, and may be human or of other species, e.g. murine, preferably human. Alternatively, monocyte cul~liLiul ,e~ medium may be used as a source of cytokines (see for example, O'Doherty et al. (1994) supra.). The amount of the cytokines will generally be in the range of about 1 ng/ml to 1 llg/ml.
Appropriate culture conditions may be ~Illpilic~'ly tested by assaying the resulting cells for their ability to present antigen. Various methods of 3s determining antigen ~l~sellLillg activity are known in the art and may be used for this purpose. For example, the ability of cells to induce ~lulife~dliu~ of freshly isolated T cells stimulated with sub-optimal collce"Lldlic.,)s of anti-CD3 ~ WO96128732 2 1 90 3 ~ 7 P~ 6~
may be measured (see Thomas et ar. (1994), supra.). In most cases, increased ability to present antigen is found after culture, frequently acc~",,ùa"ied by an increase in the e~ ssio,~ of B7 (CD80, CD86).
The enriched population of dendritic cells, either before or after culture, s may be used as antigen ,., ~s~"li"g cells to prime T cells in vivo or in vi~ro. The cells are combined with a protein antigen, or with a peptide thereof. Antigenic peptides will usually be from about 6 to 20 amino acids in length, more usually from about 10 to 18 amino acids. The peptides may have a sequence derived from a wide variety of proteins. In many cases it will be desirable to use peptides which act as T cell epitopes, usually immunodominant sequences.
The epitopic sequences from a number of antigens are known in the art.
Altennatively, the epitopic sequence may be e",,ui,iu~lly determined, by isolating and sequencing peptides bound to native MHC proteins, by synthesis of a series of peptides from the target sequence, then assaying for T cell reactivityto the different peptides, or by producing a series of binding cu,,,ule~es with different peptides and quantitating the T cell binding. Pl~pdldliol~ of fragments, identifying sequences, and identifying the minimal sequence is amply described in U.S. Patent No. 5,019,384, iss. 5-28-91, and ,~re,t,nces cited therein. The peptides may be prepared in a variety of ways as known in the art.
Antigens of interest include tumor cell antigens, allogeneic MHC
antigens, allergens, proteins of pd~l,oge,~ic Ol~dll;~lllS, including viruses, e.g.
HIV-1, hepatitis, herpesviruses, enteric viruses, respiratory viruses, rhabdovirus, rubeola, poxvirus, paramyxovirus, morbillivirus, filovirus, etc.
Infectious agents of interest also include bacteria, such as Pneumococcus, Staphylococcus, Bacillus, Str~ptococcl~s, Meningococcus, Gonococcus, Eschericia, Klebsiella, Proteus, Pseudomonas, Salmonella, Shigella, Hemophilus, Ye~sinia, Listeria, Cory"eba~ rium, Vibrio, Clostridia, Chlamydia, Mycobacterium, Helicobacterand Treponema; ,~llUIO~Udll pathogens, and the like.
Dendritic cells are pulsed in vitro with antigens by placing the cells in a phy~i~lo~ .'!y ~,c,~ le buffer containing antigen at a col1ce,,L,d~iull from at least about 0.1 IlM to as much as about 1 mM. Peptide antigens will typically be effective at a lower ~,~ce"~,dlio~ than intact protein antigens, or cell Iysates.
The cells will be incubated with antigen, generally at 37 C, for a period of time sufficient to bind the antigen to the cell. Peptide antigens will usually be incubated for at least about 1 hour, and for as long as 6 hours or more. Intact W096/28732 21 qO367 r~ ,.,,6~^~?~ ~
protein antigens will usually be incubated for at least about 3 hours, and for as long as about 12 hours or more.
The antigen pulsed DCs may be used to stimulate a T cell response against the antigen. The T cells may be in vivo, either an autologous or ~ eiG host, or may be an in vitro culture. For in vivo use the dendritic cells may be administered in any physiolo~ir~lly acceptable medium, by sub-cutaneous, intravenous, intra-dermal, etc a~ lia~ld~ioll. Usually, at least 1x104 cells will be a~",i"i~le,c:d, preferably 1x105 or more. The cells may be introduced by injection, catheter, or the like. If desired, d~per,di"g upon the purpose of the introduction of the cells, factors may also be included, such as the interleukins, e.g. IL-2 and IL-1, as well as the other interleukins, the colony stimulating factors, such as G-, M- and GM-CSF, i"lerre,u"s, e.g. ~-interferon, er~ll,,u~,oit:li,,, etc. The amount of these various factors will depend upon the purpose of the a~l" Ii:~ldliUII of the cells, the particular needs of the patient, 1S and will normally be dt:l~""i"ed ~ Jilic.~l'y.
The antigen pulsed dendritic cells or membranes thereof may be used as imm~"oacls~, bdl 1~:~ to obtain antigen specific T cells. Quantitation of T cells may be performed to monitor the p~uu~sion of a number of conditions assoc;dled with T cell activation, including autoimmune diseases, graft rejection, viral infection, bacterial and protozoan infection.
The following examples are offered by way of illustration and not by way of limitation.
EXPERIMENTA
FY~mDle 1 2s Purification of CD3-. CD14-. CD16-. CD19-. HLA-DR+ Dendritic Cells by HGMS
Materials and Methods Preparation of human NPBC. NPBC were obtained from leukocyte-rich buffy coats by centrifugation over Ficoll-Hypaque (Pharmacia, Uppsala, Sweden). After centrifugation, interphase cells were collected, resuspended in buffer and sedimented at 300 x 9 and then once again resuspended in buffer and centrifuged at 200 x 9 to remove platelets.
Labeling. About 2 x 108 NPBC were incubated with biotinylated a-CD3 (5 llg/ml), rx-CD14 (2 Ilg/ml), a-CD16 (2 ~Lg/ml), -CD19 (5 ,ug/ml), digoxigenin-conjugated anti-HLA-DR monoclonal antibody (mAb) (5 llg/ml) and human IgG
3s (1.6 mg/ml) for 10 minutes at 8C in a volume of 1 ml PBS, 1% bovine serum albumin, and then washed twice (300 x 9).

1-- wo 96/28732 2 1 9 0 3 6 7 PCT/llS96103265 Magnetic Labeling. Cells labeled with biotinylated antibodies as described above were incubated with streptavidin-conjugated colloidal su,oe,,udrdlllagnetic microparticles at 8C in a volume of 1 ml. After 15 minutes, streptavidin-CyChrome (Pharmingen, San Diego, CA) was added (2 Lg/ml) and the cells were incubated for another 5 minutes at 8C. The cells were then washed again and resuspended in 1 ml of buffer.
Depletion of Cells by HGMS. M ~ Liudll; labeled CD3, CD14, CD16 and CD19 positive cells were removed by passage over a CS column (magnetizable steelwool matrix) inserted in a MACS pe""a,)~"l magnet and sorted essentially as described by Miltenyi et al. (1992) supra. For optimal depletion, the flow rate was kept to app,u,.i"...",ly 3 ml/min. by using a 22G
needle at the outlet of the MACS column. The cells were then washed off with 1 ml of buffer. To evaluate the efficiency of the cell sepa,dlio,), aliquots of ~IISepdldlt:d cells, and from the magnetic and nonmagnetic cell fractions were analyzed by two parameter cytometric analysis with a FACScan flow cytometer (Becton Dickinson, San Jose, CA). 10,000 events per sample were recorded and analyzed using FACScan research software (Becton Dickinson). Live cells were gated according to scatter and relative propidium iodide to phycoerythrin stainin3, with propidium iodide (Pl) sl,eci~i 'Iy staining for dead cells.
Anti-Llis~oxigenin Labeling. NPBC depleted of CD3, CD14, CD16 and CD19 positive cells were incubated with anti-di~uxige,)i" monoclonal antibody conjugated to colloidal superparamagnetic ,,,i~,,uua,liules at 8C in a volume of 1 ml. After 15 minutes, anti-di~u,~ige"i" monoclonal antibodies conjugated to PE were added, and the cells were incubated for an additional 5 minutes at 80C The cells were then washed again, and resuspended in 0.5 ml of buffer.
E~r~ of Cells by HGMS Mau"t:~ic~''y labeled HLA-DR+ cells were enriched on a MiniMACS column (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany) inserted in a MiniMACS permanent magnet. Negative cells were washed off the column at flow rates of approx. 0.35 ml/min. in a volume of 1 ml.The column was then washed again four times with 0.5 ml of buffer. Finally, the HLA-DR+ cells were eluted from the column outside of the magnet in 1 ml of PBSIBSA. The number of live cells was evaluated just before and after loading the MACS column in the depletion and e,)riul"n~"~ step by a Neubauer counter chamber. Dead cells were excluded by trypan blue staining. To evaluate the efficiency of the cell sepd,d~io,), aliquots of ~lls~d,dl~d cells, and from the magnetic and non-magnetic cells fractions were analyzed by flow cytometry using a FACScan.
Counterstaining. Freshly isolated dendritic cells from peripheral blood were incubated with either a-CD4-FlTC, a-CD11c-FlTC, -CD33-FITC or a-s HLA-DR for 10 minutes at 8 C. Afterwards, cells were washed once and then analyzed by flow cytometry.
Dendritic Cell Culture. Dendritic cells were cultured in complete RPMI
1640 medium Collld;"i"g serum, penicillin, streptomycin, glutamine and 5%
FCS for 70 hours.

The analysis of the sorted populations is shown in Figure 1. A dendritic cell population was isolated with 95% purity, having the phenotype of CD3-, CD14-, CD16-, CD19-, HLA-DR+. Dendritic cells were CD4 positive. Two different populations were present, and could be distinguished on the basis of CD11c e~,u,t:ssiull. Dendritic cell precursors are CD11c negative, and mature dendritic cells are CD11c positive. Freshly isolated dendritic cells lack the ~,I,a,~ul,~ lic dendritic Illul,ul~ology, and have the d,upedldl1c~ of medium sized cells with a slightly higher forward scatter than Iymphocytes.
Figures 1A to 1C show the results of flow cytometric ",o,)iL~,i"g of the magnetic purification procedure. CD3-, CD14-, CD16-, CD19- HLA-DR~ DC
were enriched from fresh NPBC by immunomagnetic depletion of CD3, CD14, CD16 and CD19 positive cells and sllhseq~ t en,i..l""~"l of HLA-DR positive cells. CD3, CD14, CD16 and CD19 positive cells were indirectly ",ay"~Lic.l'y 2s labeled using biotinylated monoclonal a"~iL~odi~s and streptavidin-conjugated colloidal superparamagnetic mic,upd,Licles. HLA-DR positive cells were indirectly labeled using a digoxigenin coupled anti-HLA-DR mAb and ~i~u~i~el,i" mAb coupled to colloidal supe,~d,d",a~"etic particles. Cells were stained for CD3, CD14, CD16 and CD19 using streptavidin CyChrome, and for 30 HLA-DR using anti-diyu,ci~t:"i" mAb coupled to phycoerythrin. Live cells weregated according to light scatter signals. The pel.:e"~ag~ of dendritic cells is shown beside the boxed cells.
Figure 1A(i) is a dotplot of CyChrome anti-CD3; CD14; CD16 and CD19 staining vs. PE anti-HLA-DR staining of ul~sepdldled NPBC. Figure 1A(ii) is a 3s dotplot of forward scatter (FSH-H) and side scatter (SSH-H) signals from the same population as (i). Figure 1 B(i) shows NPBC after magnetic depletion of W096128732 2 1 9 0 3 6 7 PCT/US96/0326s CD3, CD14, CD16 and CD19 positive cells. Figure 1B(ii) is the forward and side scatter from the same population as (i). Figure 1C(i) shows NPBC after depletion of CD3, CD14, CD16 and CD19 positive cells and er"i~,l""t:"l for HLA-DR+ cells. Figure 1C(ii) is the forward and side scatter from the same 5 population as (i).
Figure 2A is a dotplot of CD3- CD14- CD16- CD19- HLA-DR' dendritic cells after cou"le,~l&;.,;"g with CD4-FITC. Figure 2B shows the same population as (2A) after CoullL~Ialdillillg with CD11c-FlTC. Figure 2C shows a cu",pa(i~o,) of CD33 staining for ull~epdldled pe,i~,h~,dl blood mononuclear 0 cells and for the isolated dendritic cell population shown in (2A). Among ull~epa~dled cells, CD33 is ~ sed on monocytic cells. Expression of CD33 on dendritic cell precursors is weaker than that seen with monocytes;
with mature dendritic cells it is stronger. Figure 2D shows a co"lpd,ison of CD11b staining for unse~d,dled peripheral blood mononuclear cells and for 15 the isolated dendritic cell population shown in (2A). Among unseparated peripheral blood mononuclear cells, CD11b is ~ ssed at high levels on monocytes but in lesser amounts on NK cells. Expression on dendritic cells is CUlll~dl dble to NK cells, but weaker than that seen with monocytes.
Figure 3 is a dotplot of the scatter signal from CD3- CD14- CD16- CD19-20 HLA-DR ' dendritic cells after a culture period of 70 hours. As compared to the freshly isolated ceils, shown in Figure 1C(ii), there is a siU"iri.,a"l increase in the side scatter.
Freshly isolated dendritic cells lack the characteristic dendritic Illo,~,l,ology and have the d~ped,c",ce of medium sized cells with a slightly 2s higher forward scatter than Iymphocytes. One can distinguish between round cells with simple oval or inented nuclei and less round ceils with mildly ruffled borders and more c~"" ' ' ' or lobulated nuclei. After a period of culture, blood DCs develop the typical DC Illul~ ology, exhibiting dendritic pruc~sses.
Fx~mple 2 Purification Df CD3- CD14-, CD16- CD4' Dendritic Cells by HGMS
The materials and methods were essellLi~'ly as described for Example 1, with the following cli,rel~,lces. 2 x 108 freshly isolated peripheral blood mononuclear cells were incubated with biotinylated a-CD3 (5 llg/ml), -CD14 3s (2 Ilg/ml) and a-CD16 (2 Ilg/ml); and diuuxige~ -conjugated a-CD4 mAb (2 Ilg/ml) and human IgG (1.6 mg/ml) for 10 minutes at 8C in a volume of 1 ml, and then washed twice (300 x 9). The s~hce~lu~nt immunomagnetic depletion 1s lVO 96128732 2 ~ 9 3 3 6 7 PCTIUS96/03265 ~
step selected for cells lacking CD3 CD14 and CD16. The e"ri- l""~"l step selected for cells expressing CD4.
The analysis of the sorted populations is shown in Figure 4. A dendritic cell population was isolated with 97% purity having the phenotype of CD3-CD14- CD16- and CD4+. Figures 4A to 4C show the results of flow cytometric ~o~ lillg of the magnetic purificatlon procedure. CD3- CD14- CD16- and CD4+ DC were enriched from fresh NPBC by immunoma3netic depletion of CD3 CD14 and CD16 positive cells and s~hseq~lent enrichment of CD4 positive cells. CD3 CD14 and CD16 positive cells were indirectly ",a~"~ a J
labeled using biotinylated Illul~ocl.llldl a"lil,odies and streptavidin-conjugated colloidal su~.e,~ald",ay"~ ,upd,licles. CD4 positive calls were indirectly labeled using a ~ o~igeni" coupled anti-CD4 mAb and di~o~ige"i" mAb coupled to colloidal su~ dldllla~ particles. Cells were stained for CD3 CD14 and CD16 using streptavidin CyChrome and for CD4 using anti-liyu,d~e"i" mAb coupled to phycoerythrin. Live cells were gated according to light scatter signals. The pe,~ enldge of dendritic cells is shown beside the boxed cells.
Figure 4A(i) is a dotplot of CyChrome anti-CD3; CD14 and CD16 staining vs. PE anti-CD4 staining of u llst:~Jdldled NPBC. Figure 4A(ii) is a dotplot of forward scatter (FSH-H) and side scatter (SSH-H) signals from the same population as (i). Figure 4B(i) shows NPBC after magnetic depletion of CD3 CD14 and CD16 positive cells. Figure 4B(ii) is the forward and side scatter from the same population as (i). Figure 4C(i) shows NPBC after depletion of CD3 CD14 and CD16 positive cells and enrichment for CD4+
2s cells. Figure 4C(ii) is the forward and side scatter from the same population as (i) Figure 5 is a dotplot of CD3- CD14- CD16- CD19- CD4+ dendritic cells after cou"le,~ ;.,i"g with a-HLA-DR. Almost all cells isolated according to e,~ ssio" of CD4 express HLA-DR.
It is evident from the above results that the subject invention provides for a simple fast method for sepdldli"~ l1ellldL~ lic dendritic cells and dendritic precursor from blood. The ease of operation and ability to scale up the number and size of samples provide si~"irica,1L benefits over existing methods. The cells are useful as a source of antigen ~,t,se"li"g cells for in vivo and in vitro T cell stimulation. The cells are also useful in analysis of dendritic cell growth ~irrt,e "~idli~." and function.

W096/28732 2 ~ 90367 r~ C
All p~ 5 and patent ~ s cited in this s~,eciri.,aLioll are herein i"cc"l,o,dLed by reference as if each individual pu' ': ' - 1 or patent l were :",eciri,. 'Iy and individually indicated to be incorporated by reference.
- 5 Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity of ~" ,de, bla,~i"~, it wiil be readily apparent to those of ordinary skill in the art in light of the teachings of this invention that certain changes and modiri~,dLiolls may be made thereto without departing from the spirit or scope of the appended claims.

Claims (13)

WHAT IS CLAIMED IS:

1. A method for enrichment of hematopoietic dendritic cells from a blood sample the method comprising:
preparing a suspension of nucleated cells from said blood sample;
adding to said suspension of nucleated cells magnetically coupled reagents specific for one or more cell surface antigens expressed by non-dendritic hematopoietic cells and absent on dendritic cells;
passing said suspension of cells through a ferromagnetic matrix in the presence of a magnetic field;
collecting cells that are unbound to said ferromagnetic matrix to provide a depleted sample substantially free of cells comprising said one or more cell surface antigens expressed by non-dendritic hematopoietic cells and absent on dendritic cells;
adding to said depleted sample magnetically coupled reagent specific for a cell surface antigen expressed by dendritic cells;
passing said depleted sample through a ferromagnetic matrix in the presence of a magnetic field;
washing said matrix of unbound cells; and eluting bound cells from said matrix in the substantial absence of said magnetic field to provide an enriched cell sample comprising hematopoietic dendritic cells.

2. A method according to Claim 1, wherein said enriched cell sample comprising hematopoietic dendritic cells comprises at least 95%
hematopoietic dendritic cells.

3. A method according to Claim 2, wherein said cell surface antigen expressed by dendritic cells is CD4 or an HLA class II protein.

4. A method according to Claim 3, wherein said HLA class II protein is HLA-DR.

5. A method according to Claim 2, wherein said magnetically coupled reagent specific for a cell surface antigen expressed by dendritic cellsis specific for a cell surface antigen expressed by mature hematopoietic dendritic cells.

6. A method according to Claim 5 wherein said cell surface antigen expressed by mature hematopoietic dendritic cells is CD33 CD11c or CD45RA.

7. A method according to Claim 2 wherein said magnetically coupled reagent specific for a cell surface antigen expressed by dendritic cellsis specific for a cell surface antigen expressed by precursor hematopoietic dendritic cells.

8. A method according to Claim 7 wherein said cell surface antigen expressed by precursor hematopoietic dendritic cells is CD45RO.

9. A method according to Claim 2 wherein said one or more cell surface antigens expressed by non-dendritic hematopoietic cells and absent on dendritic cells are CD3 and one of CD14 and CD11b.

10. A method according to Claim 9 wherein said one or more cell surface antigens expressed by non-dendritic hematopoietic cells and absent on dendritic cells further comprise at least one of CD16 and CD19.

11. A method for enrichment of antigen presenting cells the method comprising enriching for a population of hematopoietic dendritic cells according to Claim 1;
adding said enriched population to a liquid culture medium and growing in vitro for at least one day.

12. A method according to Claim 11 wherein said liquid culture medium comprises the cytokine GM-CSF at a concentration sufficient to enhance the differentiation of precursor hematopoiethic dendritic cells to mature dendritic cells.

13. A kit for separation of dendritic cells from blood, comprising:
two columns of a ferromagnetic matrix;
a cocktail of magnetically coupled antibody specific for CD14 and CD3;
magnetically coupled antibody specific for at least one of CD4 and HLA-DR.